ABSTRACT Mycoplasmas are significant pathogens with unique features such as the lack of a cell wall and are dependent on a host to provide essential nutrients such as cholesterol. The genomes of most species such as the human pathogen Mycoplasma pneumoniae and the murine pathogen Mycoplasma pulmonis code for about 600-700 proteins. Despite the limited proteome, mycoplasmas synthesize rhamnose and produce glycolipids, glycoproteins, and polysaccharides. Mycoplasmas have many traditional lipoproteins with the lipid attached to a cysteine residue at the amino terminus. We have shown that these lipoproteins are heavily glycosylated with glucose. Rhamnose is not attached to the lipoproteins but instead is attached to other proteins that can be associated with the membrane. An example is the glycolytic enzyme enolase, which is usually cytoplasmic but can moonlight on the surface of many organisms. We have shown that soluble enolase lacks a post-translational modification (PTM) but membrane-enolase is attached to lipid via a rhamnose linker. We find that enolase and several other proteins that are known to moonlight in bacterial membranes are released from the mycoplasmal cell surface by digestion with phospholipase. We propose to examine in aim 1 the site of rhamnosylation in proteins that are released from cells by phospholipase to identify an amino acid consensus sequence for rhamnosylation. In aim 2 the enolase gene of M. pulmonis will be engineered such that the amino acid to which rhamnose is attached will have been changed to an alternative amino acid that cannot be glycosylated. The mutated enolase gene will be inserted into the mycoplasma genome, and the effect of the mutation on protein trafficking will be examined. Other amino acids in the consensus sequence will also be mutated to identify which amino acids are important. Aim 3 is a short aim in which enolase in the walled bacterium Streptococcus pneumoniae will be examined to determine whether it possesses a PTM as found for mycoplasma. The results will yield new and valuable insight into mechanism of trafficking of moonlighting proteins.